Protocol for Detection of Bacillus anthracis in ...

Protocol for Detection of Bacillus anthracis in Environmental Samples During the Remediation Phase of an Anthrax Incidentwww.epa.gov/homeland-security-research
Protocol for Detection of Bacillus anthracis in Environmental Samples During the Remediation Phase of an Anthrax Incident Second Edition
SCIENCE
Homeland Security Research Program
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EPA/600/R-17/213
Environmental Samples During the
Remediation Phase of an
Cincinnati, OH 45268
Office of Research and Development
Homeland Security Research Program
ii
Disclaimer
The U.S. Environmental Protection Agency through its Office of Research and Development funded and
managed the effort on the protocol described here under a contract (EPA EP-C-15-012) to CSRA. The
contractor role did not include establishing Agency policy. This protocol has been subjected to the
Agency’s review and has been approved for publication. Note that approval does not signify that the
contents necessarily reflect the views of the Agency. Mention of trade names, products, or services does
not convey official EPA approval, endorsement, or recommendation.
Questions concerning this document or its application should be addressed to:
Sanjiv R. Shah, Ph.D.
U.S. Environmental Protection Agency
1300 Pennsylvania Avenue, NW
iii
4.0 Safety ............................................................................................................................................... 5
5.0 Supplies and Equipment................................................................................................................ 6
5.2 Supplies for Real-time PCR Method Based Sample Analysis ................................................. 8
5.3 Supplies for RV-PCR Method Based Sample Analysis ........................................................... 8
5.4 Supplies for Culture Method Based Sample Analysis ............................................................. 8
5.5 Equipment ................................................................................................................................... 9
9.1 Sample Processing: Spore Recovery........................................................................................ 17
9.3 Real-time PCR Analyses ........................................................................................................... 27
10.0 Rapid Viability-Polymerase Chain Reaction (RV-PCR) Method ........................................... 32
10.1 RV-PCR ..................................................................................................................................... 32
10.3 RV-PCR Sample Processing: Buffer Washes and Broth Culture ........................................ 43
10.4 Manual DNA Extraction and Purification Using the MagneSil® Blood Genomic, Max
Yield System, Kit ...................................................................................................................... 44
10.5 Real-time PCR Analysis of T0 and T9 or Tf DNA Extracts .................................................... 48
Protocol for Detection of Bacillus anthracis in Environmental Samples
iv
11.1 Sample Processing and Plating for Sponge-Sticks and Wipes .............................................. 49
11.2 Sample Processing and Plating for Swabs .............................................................................. 53
11.3 Sample Processing and Plating for Air Filters ....................................................................... 56
11.4 Sample Processing and Plating for 37-mm Filter Cassettes and Filters .............................. 59
11.5 Sample Processing and Plating for Water Samples ............................................................... 63
11.6 Confirmation of B. anthracis Colonies by Real-time PCR Analysis ..................................... 66
12.0 Data Analysis and Calculations .................................................................................................. 68
12.1 Real-time PCR Analysis ........................................................................................................... 68
12.2 RV-PCR Analysis ...................................................................................................................... 69
12.3 Culture Analysis ........................................................................................................................ 70
13.0 Method Performance ................................................................................................................... 72
14.0 Pollution Prevention .................................................................................................................... 72
14.0 Waste Management ..................................................................................................................... 72
15.0 References ..................................................................................................................................... 73
Appendix A Concentration of Water Samples using Ultrafiltration (UF) for the Detection of
Bioterrorism Threat (BT) Agents in Potable Water Samples ................................................ A-1
v
Table 2. Example Working Stock Preparation ............................................................................................ 12
Table 3. Sample Processing Negative Controls .......................................................................................... 16
Table 4. Example EPA-2 Single-plex PCR Master Mix Preparation for 70 Reactions .............................. 30
Table 5. Example EPA-1 or BC3 Single-plex PCR Master Mix Preparation for 70 Reactions ................. 30
Table 6. PCR Cycling Conditions ............................................................................................................... 31
List of Figures
Figure 1. Sample processing and spore recovery steps for real-time PCR. ................................................ 18
Figure 2. Real-time PCR amplification ....................................................................................................... 28
Figure 3. Example real-time PCR amplification curves for the initial T0 aliquot and the Tf (Final)
endpoint aliquot. ......................................................................................................................... 33
Figure 4. Flow chart for RV-PCR sample analysis. .................................................................................... 35
Figure 5. Sample processing and spore recovery steps for RV-PCR. ......................................................... 37
Figure 6. B. anthracis colonies on SBA. ..................................................................................................... 52
vi
Acronyms
BHI Brain heart infusion
BSC Biological safety cabinet
CFR Code of Federal Regulations
CFU Colony forming unit
HCL Hydrochloric acid
IEC International Electrotechnical Commission
LRN Laboratory Response Network
MCE Mixed cellulose ester
NIST National Institute of Standards and Technology
NG No growth
ORD Office of Research and Development
OSHA Occupational Safety and Health Administration
oz. Ounce
PC Positive control
PPE Personal protective equipment
vii
PT Proficiency testing
QA Quality assurance
QC Quality control
SBA Sheep blood agar
SDS Safety data sheet
SOP Standard operating procedure
SWGFACT Scientific Working Group on Forensic Analysis of Chemical Terrorism
T0 Time zero (no incubation)
T9 Nine-hour incubation
TE Tris(hydroxymethyl)aminomethane- hydrochloric acid -EDTA
TNTC Too numerous to count
TSB Trypticase™ soy broth
viii
Amicon® MilliporeSigma Corporation Billerica, MA
Applied Biosystems® Thermo Fisher Scientific Inc. Carlsbad, CA
Autovial™ Whatman™ Ltd. Maidstone, United Kingdom
BBL™ BD Diagnostics Corporation Sparks, MD
Biopur® Safe-lock® Eppendorf NA United States
Black Hole Quencher® Biosearch Technologies, Inc. Novato, CA
Clay Adams™ BD Diagnostics Corporation Sparks, MD
Cole Parmer® Cole Parmer® LLC Vernon Hills, IL
Costar® Corning Inc. Tewksbury, MA
Dispatch® Clorox Company United States
Durapore® MilliporeSigma Corporation Billerica, MA
Dynamag™ Thermo Fisher Scientific Inc. Carlsbad, CA
Fluoropore™ MilliporeSigma Corporation Billerica, MA
GN-6 Metricel® Pall Corporation Ann Arbor, MI
Invitrogen® Thermo Fisher Scientific Inc. Carlsbad, CA
Jiffy-Jack® Cole Parmer® LLC Vernon Hills, IL
Kendall™ Covidien, Inc. Mansfield, MA
Kimwipes™ Kimberly-Clark Corporation Dallas, TX
Life Technologies™ Thermo Fisher Scientific Inc. Carlsbad, CA
MagneSil® Promega Corporation Madison, WI
Masterflex® Cole Parmer® LLC Vernon Hills, IL
MaxQ™ Thermo Scientific Inc. Lenexa, KS
MicroFunnel™ Pall Corporation Ann Arbor, MI
Nalgene® Nalge Nunc Corporation Rochester, NY
Parafilm® Bemis, Inc. Neenah, WI
Sigma-Aldrich® MilliporeSigma Corporation St. Louis, MO
Stomacher® Seward Ltd. United Kingdom
TaqMan® Thermo Fisher Scientific Inc. Carlsbad, CA
Trypticase™ BD Diagnostics Corporation Sparks, MD
Tween® MilliporeSigma Corporation St. Louis, MO
Ultracel® EMD Millipore® Corporation Billerica, MA
Ultrafree® EMD Millipore® Corporation Billerica, MA
Vacushield™ Pall Corporation Ann Arbor, MI
Velcro® Velcro Companies Manchester, NH
Versalon™ Covidien, Inc. Mansfield, MA
Whatman™ Whatman™ Ltd. Piscataway, NJ
ix
Acknowledgements
This protocol was developed under the technical leadership and direction of Sanjiv R. Shah of the
National Homeland Security Research Center (NHSRC) within the U.S. Environmental Protection
Agency’s (EPA) Office of Research and Development (ORD).
The following organizations and persons are gratefully acknowledged for their help in the preparation of
this protocol:
Centers for Disease Control and Prevention (CDC), Laboratory Response Network (LRN)
Richard Kellogg and Stephen Morse, for support and cooperation in the preparation and review of this
protocol
Laura Jevitt for providing the LRN protocols that were adapted for inclusion in this protocol
Lawrence Livermore National Laboratory (LLNL)
Staci Kane, for technical contribution and critical review of the protocol
CSRA
Yildiz Chambers-Velarde and Emily King, for technical review and editing of the protocol
Technical Reviewers
Sonia Létant (LLNL)
Douglas Anders (Federal Bureau of Investigation [FBI])
Stephanie Harris (EPA Region 10)
Jafrul Hasan (EPA Office of Pesticides Program Microbiology Laboratory)
Marissa Mullins (EPA, Office of Emergency Management)
Frank Schaefer, Gene Rice, Worth Calfee and Vincent Gallardo (EPA, ORD, NHSRC)
Quality Assurance Reviewers
Cover Photos: Left – Scanning electron micrograph of Bacillus anthracis; Right – Bacillus anthracis on
blood agar (Source: CDC/Laura Rose - Public Health Image Library)
Section 11 Figure 6: Bacillus anthracis on blood agar (Source: CDC/Megan Mathias and J. Todd Parker-
Public Health Image Library)
x
1
Introduction
The series of 2001 terrorist attacks and the anthrax bioterrorism incidents that resulted in human
casualties, and public and private facility closures, prompted enhanced and expanded national safeguards.
Multiple Presidential Directives have designated the U.S. Environmental Protection Agency (EPA) as the
primary federal agency responsible for the protection and decontamination of indoor/outdoor structures
and water infrastructure vulnerable to chemical, biological, and radiological (CBR) terrorist attacks.
Accordingly, EPA’s mission to protect human health and the environment was expanded to address
critical homeland security related needs.
The National Homeland Security Research Center (NHSRC) within the Office of Research and
Development (ORD) is EPA’s hub for providing expertise on CBR agents and for conducting research to
meet EPA’s homeland security mission needs. A focus of NHSRC’s research is to support the EPA’s
Environmental Response Laboratory Network (ERLN) and Water Laboratory Alliance (WLA), an
integrated nationwide network of federal, state, local, and commercial environmental testing laboratories.
Along with the Centers for Disease Control and Prevention’s (CDC) Laboratory Response Network
(LRN), the ERLN/WLA can be activated in response to a large-scale environmental disaster to provide
analytical capability, increase capacity, and produce quality data in a systematic and coordinated manner.
Preparedness against potential indoor or outdoor wide-area anthrax attacks is one of the highest priorities
for the ERLN/WLA. Based on the realities of response activities after the 2001 anthrax incident and
continued preparedness efforts since then, it is anticipated that during an intentional (bioterrorist attack)
or accidental release of Bacillus anthracis (B. anthracis) spores, several hundreds to thousands of diverse
environmental samples (e.g., aerosol, particulates [surface swabs, wipes, 37-mm filter cassettes and
filters, and Sponge-Sticks], air filters, and drinking water) will need to be rapidly processed and analyzed
in order to assess the extent of contamination and support the planning of decontamination efforts. A
large number of samples may also need to be analyzed to determine the efficacy of decontamination
activities during the remediation phase of the response. During an anthrax incident, EPA’s decision
makers will need timely results from rapid sample analyses for planning and assessing the
decontamination efforts. To address these critical needs, NHSRC, in collaboration with CDC and
Lawrence Livermore National Laboratory (LLNL), generated this protocol for detection of B. anthracis
spores in environmental samples.
To complement an effective sample collection strategy during a suspected B. anthracis release incident, a
systematic approach for timely and cost-effective sample analyses is critical. Such a systematic approach
also helps in effectively managing and increasing the analytical laboratory capacity. Availability of a
common analytical protocol for participant laboratories can be a significant part of such an approach.
This protocol includes three analytical methods for the detection of B. anthracis spores in various
environmental samples (e.g., aerosols [air filters], particulates [surface swabs, wipes, 37-mm filter
cassettes and filters, and Sponge-Sticks], and drinking water). To simply detect the presence of the
deoxyribonucleic acid (DNA) of B. anthracis, real-time polymerase chain reaction (PCR) based sample
analysis method is included. To detect whether viable B. anthracis spores are present in the samples,
microbiological culture and Rapid Viability-PCR (RV-PCR) analytical methods are included. This
protocol has been specifically developed for use by ERLN and WLA laboratories for the analysis of
environmental samples during an incident involving contamination from B. anthracis spores. It should be
noted that LRN laboratories providing support to EPA for environmental sample analyses may use LRN-
specific protocols.
2
Sample processing procedures are also provided for respective analytical methods for all sample types
listed earlier. Since this protocol was developed to include the analyses of diverse environmental
samples, it emphasizes appropriate sample processing as well as the DNA extraction and purification
steps to significantly remove growth and/or PCR-inhibitory materials present in the samples. This
protocol will be revised as better sample processing procedures and real-time PCR assays become
available.
For drinking water samples, large volume samples may need to be analyzed to detect low concentrations
of B. anthracis spores or vegetative cells. Therefore, the protocol also includes an ultrafiltration-based
water sample concentration procedure. For post-decontamination phase culture analyses, selected isolated
colonies will be analyzed using real-time PCR to confirm the identity of B. anthracis, as opposed to
traditional biochemical and serological testing.
Several sample processing and analysis procedures in this protocol have been derived from LRN
protocols. However, these procedures have been modified, as necessary, to address EPA’s homeland
security mission needs during the remediation phase of an anthrax incident. Therefore, these modified
procedures or this protocol itself must not be designated, referred to, or misconstrued as LRN procedures
or as an LRN protocol.
It should be noted that at the time of publication and revision, this protocol has not been validated. The real-time PCR assays included in this protocol have been only partially characterized for specificity.
These assays will be updated or replaced with fully characterized and validated assays upon availability.
During any B. anthracis related emergency situations, EPA’s use of non-validated methods in the absence
of validated methods must adhere to the EPA’s Forum on Environmental Measurement (FEM) policy
directive on method validation:
According to Agency Policy Directive FEM-2010-01, Ensuring the Validity of Agency Methods
Validation and Peer Review Guidelines: Methods of Analysis Developed for Emergency Response
Situations:
It is EPA’s policy that all methods of analysis (e.g., chemical, radiochemical,
microbiological) must be validated and peer reviewed prior to issuance as Agency
methods. There are emergency response situations that require methods to be developed
and utilized, which may or may not have previously been validated or peer reviewed
prior to use. This policy directive addresses those situations in which a method must be
developed, validated and/or peer reviewed expeditiously for utilization in an emergency
response situation. Also, in such emergency response situations only, an analytical
method may be employed that has been validated by another established laboratory
network (e.g., the Center for Disease Control and Prevention’s Laboratory Response
Network, the U.S. Department of Agriculture/Food and Drug Administration’s Food
Emergency Response Network). In those instances, the responsible federal agency will
indicate that the level of validation and/or peer review that their analytical method
underwent is consistent with the Integrated Consortium of Laboratory Networks’ (ICLN)
Guidelines for Comparison of Validation Levels between Networks. 5
The responsible
federal agency may also refer to the Validation Guidelines for Laboratories Performing
Forensic Analysis of Chemical Terrorism 6
in order for the receiving federal agency to
determine if the analytical method meets the intended purpose.
3
Any EPA regional or program office that proposes to utilize a method in an emergency
response situation is responsible for establishing and documenting to what level and by
what process the method has been validated and/or peer reviewed in accordance with this
policy. A regional or program office may determine the level of validation and/or peer
review that is necessary to provide the objective evidence that a method is suitable for its
intended purpose; however, the office must document the validation and/or peer review
information supporting use of the method. All documentation should be preserved in
accordance with the Agency’s records management policy. 5
U.S. Department of Homeland Security, Integrated Consortium of Laboratory Networks (ICLN),
ICLN Guidelines for Comparison of Validation Levels between Networks, Original Version,
http://www.icln.org/docs/sop.pdf. 6
Federal Bureau of Investigation (FBI), Scientific Working Group on Forensic Analysis of
Chemical Terrorism (SWGFACT), Validation Guidelines for Laboratories Performing Forensic
Analysis of Chemical Terrorism, Forensic Science Communications, Volume 7, Number 2, April
2005.
https://www.epa.gov/sites/production/files/2015-01/documents/emergency_response_validity_policy.pdf
Also, EPA recognizes that having analytical data of known and documented quality is critical in making
proper decisions during all phases of a response to an anthrax incident and strives to establish data quality
objectives (DQOs) for each response activity.1 These DQOs are based upon needs for both quality and
response time. EPA’s ERLN, which is tasked with providing laboratory support following homeland
security-related incidents, also has established data reporting procedures. Requirements for receiving,
tracking, storing, preparing, analyzing and reporting data are specified in the Environmental Response
Laboratory Network Laboratory Requirements Document at:
https://www.epa.gov/emergency-response/environmental-response-laboratory-network-erln-laboratory-
requirements; project-specific requirements also are included in individual Analytical Service Requests.
1 Information regarding EPA’s DQO process, considerations and planning is available at:
4
Samples During the Remediation Phase of an Anthrax Incident
1.0 Scope and Application
The purpose of this updated protocol is to provide methods that can be used to detect Bacillus anthracis
(B. anthracis) spores in environmental samples. To simply detect the presence of the deoxyribonucleic
acid (DNA) of B. anthracis, this protocol includes a real-time polymerase chain reaction (PCR) based
method. Since traditional real-time PCR methods cannot determine viability of B. anthracis spores, this
protocol includes two additional methods, Rapid Viability-PCR (RV-PCR) and culture/plating followed
by confirmation of isolate by real-time PCR. Depending upon the laboratory capability, either of these
two methods can be performed to detect viable B. anthracis spores in environmental samples. The real-
time PCR assays included in this protocol have been only partially characterized for specificity. PCR
assays included in this protocol will be updated or replaced with fully characterized and validated assays
upon availability. During an actual incident, validated assays from other sources (e.g., Department of
Defense Biological Products Assurance Office or Laboratory Response Network [LRN]) may be used.
This protocol will be periodically updated to include advances in sample processing and nucleic acid
extraction-purification procedures. This protocol is intended for the analyses of swabs, wipes, Sponge-
Sticks, 37-mm filter cassettes and filters, air filters, and water for B. anthracis.
2.0 Summary of Methods
2.1 Sample Analysis for Detection of B. anthracis DNA (Real-time PCR): Following sample
processing including DNA extraction and purification, the DNA extracts are analyzed by real-
time PCR using the Applied Biosystems® (ABI) 7500 Fast Real-Time PCR System. Direct
DNA-based analysis of samples allows for high throughput and rapid results. Unless advised
otherwise, for post-incident-recognition sample analysis and depending on the purpose of sample
analyses, real-time PCR should be performed using only the most sensitive assay, EPA-2, which
targets the capB gene (on pXO2 plasmid; Section 6.15) or an equivalent assay.
2.2 Sample Analyses for Detection of Viable B. anthracis Spores: After samples have been
appropriately processed, they are cultured by either inoculating into nutrient rich broth (Reference
15.1 [RV-PCR procedure]) or plating on sheep blood agar (Reference 15.2 [culture procedure]),
to allow for germination of viable spores and growth.
2.2.1 RV-PCR Procedure (updated)
The RV-PCR procedure serves as an alternative to the traditional culture-based methods
for detection of viable pathogens. The RV-PCR procedure integrates high-throughput
sample processing, short-incubation broth culture, and highly sensitive and specific real-
time PCR assays to detect low concentrations of viable bacterial threat agents.
Specifically, the procedure uses the change in real-time PCR response, referred to as the
change in cycle threshold, or ΔCT, between the initial cycle threshold (CT) at time 0 (T0)
(just before sample incubation) and the CT after final incubation time (Tf). Example PCR
response curves are shown in Figure 3 along with the criteria for positive detection,
namely ΔCT ≥ 9. Unless advised otherwise, for post-incident-recognition sample analysis
5
and, real-time PCR based analysis should be performed using only the most sensitive
assay, EPA-2, or all three assays included in Section 6.15, depending on the purpose of
sample analyses.
2.2.2 Culture Procedure
The culture option includes sample processing and plating serial dilutions of the
processed sample and membrane filters on a non-selective sheep blood agar (SBA)
followed by rapid confirmation of typical isolated colonies using B. anthracis specific
real-time PCR. Unless advised otherwise, for post-incident-recognition sample analysis,
real-time PCR should be performed using only the BC3 PCR assay that targets the
marker gene on the B. anthracis genome (Section 6.15) or all three assays included in
Section 6.15, depending on the purpose of sample analyses.
3.0 Interferences and Contamination
3.1 Poor recoveries of B. anthracis spores may be caused by the presence of high numbers of
competing or inhibitory organisms, background debris, or toxic substances (e.g., metals or
organic compounds).
3.2 Metals and organic compounds may inhibit PCR reactions. After spore recovery during sample
processing, samples suspected of containing iron or rust particles should be placed on a magnetic
rack (Invitrogen® Cat. No. 123-21D or equivalent) to separate out the particulates from the
samples. The supernatant should be transferred to a clean sterile bottle or tube, using care not to
transfer any of the particulates.
3.3 Problems related to sample processing, such as clogging of filters and inefficient extraction, may
also result in poor spore recoveries.
4.0 Safety
Note: This protocol should not be misconstrued as a laboratory standard operating procedure (SOP)
that addresses all aspects of safety including biosafety while working with the Biological Select
Agents; the laboratory should adhere to safety guidelines and requirements established by their
organization or facility as well as the CDC. All wastes should be handled according to CDC &
Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition, (Reference
15.3), waste management and disposal requirements.
4.1 Safety Precautions
Direct contact of skin or mucous membranes with infectious materials, accidental parenteral
inoculation, ingestion, and exposure to aerosols and infectious droplets have resulted in B.
anthracis infection. Due to the infectious nature of this organism, all samples should be handled
and analyzed using biosafety requirements as dictated by BMBL (Reference 15.3), or the most
recent version and/or organizational health and safety plans. The CDC requires biosafety level
(BSL)-3 handling of this organism.
4.2 Additional Recommended Precautions
6
4.2.1 To the extent possible, disposable materials (e.g., pipets, loops) should be used for
sample manipulations.
4.2.2 The analyst must know and observe normal safety procedures required in a microbiology
laboratory while preparing, using and disposing of media, cultures, reagents and
materials. Analysts must be familiar with the operation of sterilization equipment.
4.2.3 Personal Protective Equipment (PPE)
Laboratory personnel processing and conducting analyses of samples for B. anthracis
must use appropriate PPE (e.g., gloves, lab coat). Also, laboratory personnel should
familiarize themselves with the specific guidance for levels of protection and protective
gear developed by the U.S. Department of Labor, Occupational Safety and Health
Administration (OSHA), as provided in Appendix B of 29 CFR 1910.120
(http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=STANDARDS&p
_id=9767). In addition to OSHA guidance, CDC developed recommendations for PPE
based on biosafety level (BSL) (Reference 15.3,
http://www.cdc.gov/biosafety/publications/bmbl5/index.htm).
Note: Remove used gloves and don new ones, as appropriate, to avoid contaminating hands
and surfaces between processing of each sample and to prevent cross-contamination.
Gloves should be disposed of (in an autoclavable biohazard bag) whenever they become
visibly contaminated or the integrity of the gloves is compromised. After all work with
potentially infectious materials is completed, gloves should be removed, properly
disposed, and hands should be washed with soap and water.
4.2.4 This protocol does not address all safety issues associated with its use. Refer to 1)
BMBL, 5th Edition, CDC 2009 (Reference 15.3) for additional safety information; 2)
organization specific Health and Safety guidelines; 3) Select Agent Program
Requirements; and 4) a reference file of Safety Data Sheets (SDS).
5.0 Supplies and Equipment
Note: Refer to Appendix A for supplies and equipment for large volume drinking water
sample processing.
5.1.2 Sterile Gloves (e.g., latex, vinyl or nitrile)
5.1.3 Bleach wipes (Dispatch® Cat. No. 69150 or equivalent)
5.1.4 Ziplock bags (large ~20” × 28” [inches], medium ~12” × 16”, small ~7” × 8”)
5.1.5 Sharps waste container
5.1.6 Absorbent pad, bench protector (Lab Source Cat. No. L56-149 or equivalent)
5.1.7 Medium and large autoclavable biohazard bags and wire twist ties
5.1.8 Sterile scalpels
7
5.1.10 Sterile disposable forceps (Cole Parmer® Cat. No. U-06443-20 or equivalent)
5.1.11 Squeeze bottle with 70% isopropyl alcohol
5.1.12 Squeeze bottle with deionized (DI) water
5.1.13 Autoclave tape
5.1.14 Autoclave bags
5.1.16 Large photo-tray or similar tray for transport of racks
5.1.17 Laboratory marker
5.1.19 Sterile disposable serological pipets: 5 mL and 50 mL
5.1.20 Sterile disposable aerosol barrier pipet tips: 1000 µL, 200 µL, 20 µL, 10 µL (Rainin Cat.
No. SR-L1000F, SR-L200F, GP-20F, GP-10F or equivalent)
5.1.21 1.5 mL Eppendorf Snap-Cap Microcentrifuge Biopur® Safe-Lock® tubes (Fisher
Scientific Cat. No. 05-402-24B or equivalent)
5.1.22 Sterile 50 mL conical tubes (Fisher Scientific Cat. No. 06-443-18 or equivalent)
5.1.23 Sterile 15 mL conical tubes (Fisher Scientific Cat. No. 339650 or equivalent)
5.1.24 Sterile 250 mL and 1 L filter systems, polyethersulfone (PES), 0.2 µm (Fisher Scientific
Cat. No. 09-741-04, 09-741-03 or equivalent)
5.1.25 Sterile 2 mL tubes, DNase, RNase-free, gasketed, screw caps (National Scientific Cat.
No. BC20NA-PS or equivalent)
5.1.26 Glass beads, acid washed, 106 µm and finer (Sigma-Aldrich® Cat. No. G4649 or
equivalent)
5.1.27 Glass beads, acid washed, 425-600 µm and finer (Sigma-Aldrich® Cat. No. G8772 or
equivalent)
5.1.28 PCR 8 cap strips (VWR Cat. No. 83009-684 or equivalent)
5.1.29 Amicon® Ultra-0.5 Centrifugal Filter Concentrator with Ultracel® 100 Regenerated
Cellulose Membrane (Millipore® Cat. No. UFC503096 or equivalent); Amicon®
collection tubes (Millipore® Cat. No. UFC50VL96 or equivalent)
5.1.30 Sterile 0.22µm Ultrafree®-MC GV 0.5 mL Centrifugal Filter Unit with Durapore® PVDF
Membrane, Yellow Color Coded (Millipore® Cat. No. UFC30GV0S or equivalent)
5.1.31 0.1 µm Ultrafree®-MC, VV Centrifugal Filter Device (Millipore® Cat. No. UFC30VV00
or equivalent)
5.1.32 Sterile wide mouth screw cap containers, 120 mL (Fisher Scientific Cat. No. 14-375-459
or equivalent)
5.1.33 Racks for 15 mL and 50 mL conical tubes
5.1.34 Sterile 2 ounce (oz., 1 ounce~30 mL) polypropylene cups with lids (Container &
Packaging Supply; Cat. No. J037 and Cat. No. L208, or equivalent)
8
5.1.35 Plastic lidded box (Fisher Scientific Cat. No. 03-484-23 with lid Cat. No. 03-484-24, or
equivalent)
5.2 Supplies for Real-time PCR Method Based Sample Analysis
5.2.1 96-well PCR plates (Applied Biosystems® [ABI] Cat. No. 4346906 or equivalent)
5.2.2 96-well plate holders, Costar®, black (VWR Cat. No. 29442-922 or equivalent)
5.2.3 Edge seals for 96-well PCR plates (Adhesive Plate Sealers, Edge Bio Cat. No. 48461 or
equivalent)
5.2.4 Foil seals for 96-well PCR plates (Polar Seal Foil Sealing Tape, E&K Scientific Cat. No.
T592100 or equivalent), for longer storage of the plates
5.2.5 Optical seals (ABI Cat. No. 4311971 or equivalent)
5.3 Supplies for RV-PCR Method Based Sample Analysis
5.3.1 30 mL screw cap tubes (E&K Scientific Cat. No. T324S or equivalent)
5.3.2 Disposable nylon forceps (VWR Cat. No. 12576-933 or equivalent)
5.3.3 Monofilament polyester mesh disc (McMaster Carr Cat. No. 93185T17 or equivalent) or
2” × 2” cut squares from mesh sheets (McMaster Carr Cat. No. 9218T13 or equivalent)
5.3.4 GE Healthcare (Whatman™) Autovial 12 Syringeless Filter™, filter vials (GSS Cat. No.
AV125NPUPSU or equivalent)
5.3.5 Pull-Tab Plug, orange cap (Caplugs Cat. No. ECP-M24 or equivalent), for vortexing and
incubation steps
5.3.6 Pull-Tab Tapered Plug, red cap (Caplugs Cat. No. CPT-10 or equivalent), to cover vial
while pipetting to prevent cross-contamination
5.3.7 Polyethylene female luer plug (Ark-Plas Products Cat. No. LPC14-PP0 or equivalent)
5.3.8 50 mL conical tubes, skirted (VWR Cat. No. 82050-322 or equivalent)
5.3.9 Disposable serological pipets: 25 mL, 10 mL, 5 mL
5.3.10 Single 50 mL conical tube holder (Bel-Art Cat. No. 187950001 or equivalent)
5.3.11 Screw cap tubes, 2 mL (VWR Cat. No. 89004-298 or equivalent)
5.3.12 96-well tube rack(s) for 2 mL tubes (8 × 12 layout) (Bel-Art Cat. No. 188450031 or
equivalent)
5.3.13 2 mL Eppendorf tubes (Fisher Scientific Cat. No. 05-402-24C or equivalent)
5.3.14 96-well 2 mL tube rack (8 × 12 format) (Bel-Art Cat. No. 188450031 or equivalent)
5.3.15 Adhesive tape (3M, Inc. Heavy Duty Scotch Tape Cat. No. 34-8711-4279-9, or
equivalent), to seal unused manifold openings.
5.4 Supplies for Culture Method Based Sample Analysis
5.4.1 Sterile disposable Petri dishes, 100 mm × 15 mm
5.4.2 Sterile disposable inoculating loops (10 µL) and needles
9
5.4.3 Sterile disposable cell spreaders (such as L-shaped, Fisher Scientific Cat. No. 03-392-150
or equivalent)
5.4.4 Sterile MicroFunnel™ Filter Funnels, 0.45 µm pore-size (VWR Cat. No. 55095-060 or
equivalent)
5.4.5 Specimen Cups, 4.5 oz. (Kendall Cat. No. 17099 or equivalent)
5.4.6 Racks for 15 mL and 50 mL centrifuge tubes
5.4.7 Sterile disposable plastic 50 mL screw cap centrifuge tubes (Becton, Dickinson and
company [BD] Cat. No. 352070 or equivalent)
5.4.8 Sterile disposable plastic 15 mL screw cap centrifuge tubes (BD Cat. No. 352097 or
equivalent)
5.4.9 Sterile pipet tips with aerosol filter for 1000 µL and 100 µL (Rainin Cat. No. SR-L1000F
and GP-100F or equivalent)
5.4.10 Biotransport carrier (Nalgene®, Thermo Scientific Cat. No. 15-251-2 or equivalent)
5.5 Equipment
5.5.1 Biological Safety Cabinet (BSC) – Class II or Class III
5.5.2 PCR preparation hood/Work station
5.5.3 Shaker incubator for RV-PCR (New Brunswick Innova 40, Eppendorf Cat. No. M1299-
0080; or Thermo Scientific, MaxQ™ 4000 Cat No. SHKE4000; or equivalent) and
Universal 18” × 18” shaker platform (New Brunswick, Cat. No. M1250-9902 or VWR,
Cat. No. 89173-848; or Thermo Scientific, MaxQ™ Cat. No. 30110; or equivalent)
5.5.4 Balance, analytical, with Class S reference weights, capable of weighing 20 g ± 0.001 g
5.5.5 ABI 7500 Fast Real-Time PCR System (Life Technologies™)
5.5.6 Refrigerated centrifuge with PCR plate adapter and corresponding safety cups and rotors
for 5 mL and 50 mL tubes (Eppendorf Cat. No. 5804R, 5810R or equivalent) or PCR
plate spinner (placed in BSC [VWR Cat. No. 89184-608 or equivalent])
Note: Swinging bucket and fixed angle rotors for the refrigerated centrifuge may also be
necessary.
Cat. No. 5415R/5424R or equivalent)
5.5.8 Filter vial manifold-top and bottom for RV-PCR (Pacon Manufacturing. Cat. No.
1701232-1 and 1701232-2 with Allen screws) and 2 top brackets (Pacon Manufacturing.
Cat. No. PART 1 with Allen screws). The bottom manifold includes an elbow port for
attaching tubing to a vacuum source.
5.5.9 T-Handle Hex Key, 6" with Cushion Grip, 9/64", Red (All-Spec Cat. No. 57306-12200 or
equivalent)
5.5.10 Capping tray for RV-PCR (Pacon Manufacturing Cat. No. 1701233)
5.5.11 30 mL tube rack for RV-PCR (Pacon Manufacturing Cat. No. 1701234)
5.5.12 Vacuum pump with gauge (Cole Parmer® Model EW-07061-40 or equivalent) or vacuum
source capable of < 10 pounds per square inch (psi) (68.95 kilopascals)
10
5.5.13 Vacuum pump filters for pump (Acrovent™ Cat. No. 4249 or equivalent)
5.5.14 Vacuum trap accessories
5.5.15 Platform vortexer (VWR Cat. No. 58816-115 or equivalent)
5.5.16 Single-tube vortexer (Fisher Scientific Cat. No. 02-215-365 or equivalent)
5.5.17 Heating block for RV-PCR (VWR Cat. No. 12621-096 or equivalent) and 2 mL tube
blocks (VWR Cat. No. 12985-048 or equivalent) or water bath set at 95°C
5.5.18 Single-channel micropipettors (1000 L, 200 L, 100 µL, 20 µL, 10 µL)
5.5.19 Serological pipet aid
5.5.20 Dynamag™ magnetic racks for RV-PCR (Invitrogen® Cat. No. 123-21D or equivalent)
5.5.21 Incubator(s), microbiological type, maintained at 37.0°C
5.5.22 Autoclave or steam sterilizer, capable of achieving 121°C (15 psi) for 30 minutes
5.5.23 Manifold incubator rack to hold up to 4 manifold/capping trays (Pacon Manufacturing
Cat. No. 1701190) and/or peg kit (Pacon Manufacturing Cat. No. 1701189-8 for New
Brunswick Innova incubator and platform or Cat. No. 1701189-10 for Thermo-Fisher
MaxQ incubator and platform) for securing individual manifold/capping trays to the
shaking incubator platform for RV-PCR
5.5.24 Cold block for 2 mL tubes (Eppendorf Cat. No. 3880 001.018 or equivalent)
5.5.25 Mini-Bead-beater (BioSpec Products, Inc. Cat. No. 607 [16 place] or equivalent)
5.5.26 Tube racks, 80-place (VWR Cat. No. 30128-282 or equivalent)
5.5.27 40 kHz sonicator bath (Branson Ultrasonic Cleaner Model 1510, Process Equipment and
Supply, Inc. Cat. No. 952-116 or equivalent)
5.5.28 Stomacher® 400 Circulator (Seward Cat. No. 0400/001/AJ or equivalent) with closure
bags (Cat. No. BA6141/CLR or equivalent) and rack (Cat. No. BA6091 [1 place] and
BA6096 [10 place] or equivalent)
6.0 Reagents and Standards
6.1 Reagent-grade chemicals must be used in all tests. Unless otherwise indicated, reagents shall
conform to the specifications of the Committee on Analytical Reagents of the American
Chemical Society (ACS) (Reference 15.4). For suggestions regarding the testing of reagents not
listed by the ACS, see AnalaR Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset,
U.K. (Reference 15.5); and United States Pharmacopeia and National Formulary 24, United
States Pharmacopeial Convention, Md. (Reference 15.6).
6.2 1X phosphate buffered saline with 0.05% Tween® 20 (PBST), pH 7.4, (Teknova Cat. No. P0201
or equivalent)
6.3 1X phosphate buffered saline (PBS), pH 7.4, (Teknova Cat. No. P0261 or equivalent)
6.4 Extraction Buffer with Tween® 20 for RV-PCR
6.4.1 Composition:
11
200 proof ethanol 300 mL
6.4.2 Add 1X PBS 0.05% Tween® 20 to ethanol and mix well. Filter sterilize using a 1 L, 0.22
µm PES filtering system with disposable bottle. Store solution at 4°C until time of use
for a maximum of 90 days.
6.5 Extraction Buffer without Tween® 20 for RV-PCR
6.5.1 Composition:
200 proof ethanol 300 mL
6.5.2 Add 1X PBS to ethanol and mix well. Filter sterilize using a 1 L, 0.22 µm PES filtering
system with disposable bottle. Store solution at 4°C until time of use for a maximum of
90 days.
6.6 High Salt Wash Buffer (10X PBS, pH 6.5, Teknova Cat. No. P0185 or equivalent) for RV-PCR
6.7 Low Salt Wash Buffer (1X PBS, pH 7.4, Teknova Cat. No. P0261 or equivalent) for RV-PCR
6.8 70% Ethanol for RV-PCR − Aseptically mix 70 mL of ethanol (100%) with 30 mL of sterile
PCR-grade water. Dispense into 2-3, sterile 50 mL conical tubes and store at 4°C, for a
maximum of one week. Unopened tubes may be stored for up to one month at 4°C.
6.9 PCR-grade water, sterile (Teknova Cat. No. W3350 or equivalent)
6.10 0.1 M sodium phosphate/10 mM EDTA (Ethylenediaminetetraacetic acid) buffer/0.01% Tween®
20, pH = 7.4 (Teknova Cat. No. S2216 or equivalent)
6.11 TE buffer (1X Tris [10 mM]-HCl-EDTA [1 mM]) buffer, pH 8.0 (Fisher Scientific Cat. No.
BP2473-500 or equivalent)
6.12 Promega reagents for DNA extraction and purification procedure for RV-PCR:
MagneSil® Blood Genomic, Max Yield System, Kit (Promega Cat. No. MD1360; VWR Cat.
No. PAMD1360)
MagneSil® Paramagnetic Particles (PMPs) (VWR Cat. No. PAMD1441)
Lysis Buffer (VWR, Cat. No. PAMD1392)
Elution Buffer (VWR Cat. No. PAMD1421)
Alcohol Wash, Blood (VWR Cat. No. PAMD1411)
Anti-Foam Reagent (VWR Cat. No. PAMD1431)
6.13 TaqMan® Universal PCR Master Mix (Thermo Fisher/Life Technologies, Cat. No. 4304437)
6.14 Platinum® Taq DNA Polymerase (Thermo Fisher/Life Technologies, Cat. No. 10966034)
6.15 PCR Assays
EPA-2 PCR assay targeting the capB gene on the pXO2 plasmid (Reference 15.7)
Forward Primer (BA-EPA-2F) – 5’-TGCGCGAATGATATATTGGTTT-3’
12
Probe (BA-EPA-2Pr) – 5’-6FAM-TGACGAGGAGCAACCGATTAAGCGC-BHQ1-3’
EPA-1 targeting the pagA gene on pXO1 plasmid (Reference 15.7)
Forward Primer (BA-EPA-1F) – 5’-GCGGATAGCGGCGGTTA-3’
Reverse Primer (BA-EPA-1R) – 5’-TCGGTTCGTTAAATCCAAATGC-3’
Probe (BA-EPA-1Pr) – 5’-6FAM-
ACGACTAAACCGGATATGACATTAAAAGAAGCCCTTAA-BHQ1-3’
BC3 targeting a hypothetical gene on the chromosome of B. anthracis
Forward Primer (BA-BC3-F) – 5’-TTTCGATGATTTGCAATGCC-3’
Reverse Primer (BA-BC3-R) – 5’-TCCAAGTTACAGTGTCGGCATATT-3’
Probe (BA-BC3-Pr) – 5’-6FAM-ACATCAAGTCATGGCGTGACTACCCAGACTT-BHQ1-
6.15.1 Preparation of concentrated and primer and probe working stocks
Prior to PCR analyses lyophilized primers and probes should be rehydrated in PCR-grade
water to prepare concentrated stocks. Primary concentrated storage stocks should
initially be prepared to obtain 100 μM (0.1 nmoles/μL) and 20 μM (0.02 nmoles/μL)
solutions of primers and probes, respectively. These primary (concentrated) stocks will
be used to prepare working stock solutions which will then be used to prepare PCR assay
mixes (Section 9.3.3) on the day of use. Examples of rehydration of lyophilized
primers/probes and dilution of rehydrated stocks to prepare working stocks are provided
in Tables 1 and 2, respectively.
Table 1. Example Concentrated Stock Preparation
Lyophilized Primer/Probe
Probe 10 500 0.02 20
Table 2. Example Working Stock Preparation
Concentrated Stock
Probe 20 180 0.1 0.002 2
Working stocks will be used to prepare master mix on the day of use (Section 9.3.3).
6.16 Positive Control (PC) –DNA isolated from an appropriate virulent B. anthracis strain containing
all of the plasmids (pXO2, pXO1). For culture analyses, B. anthracis Sterne (BSL-2 organism)
or other avirulent strains may be used as a PC to meet the laboratory’s BSL.
6.17 Trypticase™ Soy Agar with 5% Sheep Blood (SBA)
13
6.17.1 The use of commercially prepared SBA plates is recommended (VWR Cat. No. 90001-
276 or 90001-282 or equivalent), however dehydrated medium (BBL™ Cat. No. 227300
or equivalent), with the addition of sheep blood (Oxoid Cat. No. SR0051 or equivalent)
may be used. If commercially prepared medium is not available, prepare medium using
procedures in Sections 6.17.2-6.17.4.
6.17.2 6.17.2 Medium Composition:
Tryptone H 15 g
Sheep blood 50 mL
Reagent-grade water ~900 mL
6.17.3 Add reagents except sheep blood to 850 mL of reagent-grade water and mix thoroughly
using a stir bar and hot plate. Boil for 1 minute with rapid stir bar agitation to dissolve
completely. Adjust pH to 7.3 ± 0.2 with 1.0 N HCl or 1.0 N NaOH and bring to 950 mL
with reagent-grade water. Autoclave at 121°C (15 psi) for 15 minutes. Do not overheat.
Cool to 45°C-50°C in a water bath.
6.17.4 Aseptically add 50 mL of sterile sheep blood (5.0% final concentration) to the cooled
medium and mix well. Aseptically pour 12-15 mL medium into each 100 mm × 15 mm
sterile Petri dish. After agar solidifies, store at 4°C for a maximum of two weeks.
6.18 Brain Heart Infusion Broth for RV-PCR (BHI broth)
6.18.1 The use of commercially prepared medium is recommended (Fisher Scientific Cat. No.
DF0037-15-0 or equivalent), however dehydrated medium (BBL™ Cat. No. 237500 or
equivalent), may be used. If commercially prepared medium is not available, prepare
medium using procedures in Sections 6.18.2-6.18.3.
6.18.2 Medium Composition:
Proteose peptone 10.0 g
Sodium chloride 5.0 g
Dextrose 2.0 g
Reagent-grade water 1.0 L
6.18.3 Add reagents to 1 L of reagent-grade water mix thoroughly and heat to dissolve
completely. Autoclave at 121C (15 psi) for 15 minutes. Final pH should be 7.4 ± 0.2.
Store at 4°C for a maximum of three months in screw cap containers.
6.19 Trypticase™ Soy Broth (TSB)
6.19.1 The use of commercially prepared TSB is recommended (BBL™ Cat. No. 221715 or
equivalent), however dehydrated medium (BBL™ Cat. No. 211768 or equivalent), may
14
be used. If commercially prepared medium is not available, prepare medium using
procedures in Sections 6.19.2-6.19.3.
Papaic digest of soybean meal 3 g
Sodium chloride 5 g
Reagent-grade water 1.0 L
6.19.3 Add reagents to 1 L of reagent-grade water mix thoroughly and heat to dissolve
completely. Autoclave at 121°C (15 psi) for 15 minutes. Final pH should be 7.3 ± 0.2.
Store at 4°C for a maximum of three months in screw cap containers.
6.20 10% Bleach-pH amended (prepared daily), optional
Add 2 parts water to 1 part bleach, and then add 5% acetic acid (1 part) and remaining water (6
parts). Measure pH and add bleach (to increase pH) or acetic acid (to decrease pH) as needed to
obtain a final pH between 6 and 7. A pH meter, as opposed to pH strips or kit, should be used to
measure pH. When mixed, place a lid on the mixture to reduce chlorine escape and worker
exposure.
7.0 Calibration and Standardization
7.1 Check temperatures in incubators twice daily with a minimum of 4 hours between each reading to
ensure operation within stated limits. Record the temperature in a log book.
7.2 Check temperature in refrigerators/freezers at least once daily to ensure operation is within the
storage requirements for samples, reagents and media. Record daily measurements in a
refrigerator or freezer log book.
7.3 Check thermometers including those on instrumentation (e.g., digital display) at least annually
against a National Institute of Standards and Technology (NIST) certified thermometer or one
that meets the requirements of NIST Monograph SP 250-23. Check columns for breaks.
7.4 Calibrate pH meter prior to each use with at least two of three standards (e.g., pH 4.0, 7.0 or 10.0)
closest to the range being tested.
7.5 Calibrate balances once per month with reference weights (e.g., ASTM Class 2).
7.6 Micropipettors should be calibrated at least annually and tested for accuracy on a weekly basis.
7.7 Follow manufacturer instructions for calibration of real-time PCR instruments.
7.8 Re-certify BSCs annually. Re-certification must be performed by a qualified technician.
7.9 Autoclave maintenance should be conducted at least annually. Autoclave temperature and total
sterilization cycle time should be checked on a quarterly basis. Record the data in a log book.
Spore strips or spore ampules should be used monthly as bioindicators to confirm sterilization.
15
7.10 Refrigerated centrifuges should be checked to confirm temperature and revolutions per minute
(rpm) on a quarterly basis. Record the data in a log book.
7.11 Vacuum pressure (e.g., pumps, in house system) should be checked on a regular basis to ensure
that the pressure is < 10 psi. Higher or lower vacuum pressure could negatively impact
recoveries.
8.0 Quality Control (QC)
8.1 Each laboratory that uses this protocol is required to operate a formal quality assurance (QA)
program that addresses and documents instrument and equipment maintenance and performance,
reagent quality and performance, analyst training and certification, and records storage and
retrieval. International Organization for Standardization (ISO)/International Electrotechnical
Commission (IEC) 17025 (International Standard: General requirements for the competence of
testing and calibration laboratories, Section Edition 2005-05-15) provides a quality framework
that could be used to develop a formal QA program.
8.2 Sample integrity − Samples should be checked for integrity (e.g., improperly packaged,
temperature exceedance, leaking). Samples may be rejected if the integrity has been
compromised. Alternately, if sample integrity has been compromised the sample may be
analyzed and the data qualified and marked accordingly (e.g., if a sample exceeded temperature
during transport, the data would be flagged and marked as exceeding temperature), so that a
decision can made regarding whether the data should be considered valid or invalid.
8.3 Analyst qualifications − Only those analysts that have been trained and have demonstrated
proficiency with these analytical techniques should perform this procedure.
8.4 Proficiency testing (PT) − The laboratory should have analysts analyze PT samples annually at a
minimum to ensure they are maintaining proficiency. In addition, analysts should analyze PT
samples to demonstrate proficiency prior to analyzing field samples. For laboratories not
routinely using this protocol, analysts should analyze PT samples biannually. If a PT failure
occurs, the laboratory should identify and resolve any issues and then request and analyze
additional PT samples. Field samples should not be analyzed until the laboratory passes the PT.
8.5 Media sterility check − The laboratory should test media sterility by incubating a single unit (tube
or Petri dish) from each batch of medium (BHI broth, TSB and SBA) at 37ºC ± 2ºC for 24 ± 2
hours and observe for growth. Absence of growth indicates media sterility. On an ongoing basis,
the laboratory should perform media sterility checks every day that samples are analyzed.
8.6 PCR: Positive control (PC) − DNA isolated from an appropriate virulent B. anthracis strain
containing all of the plasmids (pXO2, pXO1) should be used as the PC. The laboratory should
analyze a PC in triplicate reactions with each PCR run. Prepare the PC at a concentration of 50
pg of purified B. anthracis total DNA per 5 µL of PCR-grade water. All PCs should result in a
CT ≤ 40 and replicates should be within ± 1 CT of each other.
8.7 Culture: Positive control (PC) − The laboratory should analyze PCs (known quantity of viable
spores) to ensure that all media and reagents are performing properly. B. anthracis Sterne (BSL-
2) or other avirulent strains may be used as a PC to meet the laboratory’s BSL. PCs should be
analyzed whenever a new batch of media or reagents is used. On an ongoing basis, the laboratory
should run a PC every day that samples are analyzed.
16
8.8 External inhibition control (EIC) 50 pg genomic DNA from virulent B. anthracis strain
containing all of the plasmids (pXO2, pXO1) − For determination of presence of DNA by real-
time PCR, the laboratory should analyze an EIC for each environmental sample DNA extract to
determine if the matrix is causing inhibition potentially resulting in false negative results.
Prepare the EIC at a concentration of 50 pg of purified B. anthracis DNA per 1 µL of PCR-grade
water. Using a 10 µL pipettor, carefully add 1 µL of the DNA to the EIC wells on a PCR plate
and then add 5 µL of sample DNA extract to each well and mix thoroughly. The PCR results
from the PC and EICs (both containing 50 pg of B. anthracis DNA) are then compared. Lower or
similar CT values for the EIC indicate there is no inhibition. A higher CT value for the EIC (>3
CT values) is indicative of matrix inhibition.
Note: To minimize cross contamination the EICs should not be placed next to the field samples when
setting up the PCR plate.
8.9 No template controls (NTC) − The laboratory should analyze NTCs (5 µL of PCR-grade water is
added to the NTC wells on a PCR plate in place of the DNA or the sample DNA extract) to
ensure that reagents are not contaminated. On an ongoing basis, the laboratory should analyze
NTCs in triplicate PCR reactions with each PCR run. The NTCs must not exhibit fluorescence
above the background level (i.e., no CT value). If CT values are obtained as a result of a possible
contamination or cross-contamination, prepare fresh PCR Master Mix and repeat the analysis.
8.10 Field blank − The laboratory should request that the sampling team provide a field blank with
each batch of samples. A field blank is defined as either a sample collection tool (e.g., wipe,
swab) or sterile reagent-grade water that is taken out to the field, opened and exposed to the
environment, but not used to collect a sample, and then placed in a bag and sealed and transported
to the laboratory along with the field samples. The field blank is treated as a sample in all
respects, including exposure to sampling location conditions, storage, preservation and all
analytical procedures. Field blanks are used to assess any contamination due to sampling location
conditions, transport, handling and storage. The laboratory should process and analyze this
control along with each batch of environmental samples. The field blanks should not exhibit
fluorescence (i.e., CT >45).
Note: The field blank for large volume water samples should also be concentrated using
ultrafiltration (UF) prior to analyses. A smaller volume of water (e.g., 10-20 L) may be used
for the field blank to minimize the burden on the laboratory.
8.11 Sample processing negative control (PNC) or method blank − The laboratory should process and
analyze a PNC in the same manner as a sample to verify the sterility of equipment, materials and
supplies. Absence of growth indicates lack of contamination from the target organism. Please
refer to Table 3 for appropriate PNC.
8.12 For RV-PCR based analysis, the T0 and T9 or Tf extracts are analyzed (in triplicate). PCR
positive and negative controls must be analyzed using the same preparation of the PCR Master
Mix and must be run on the same 96-well plate as the T0 and T9 or Tf extracts.
Table 3. Sample Processing Negative Controls
Matrix PNC
17
Sponge-Sticks Clean (unused) Sponge-Stick
Drinking water and decontamination
Large volume water samples 10-20 L of sterile reagent-grade water
9.0 Real-time PCR Method
Real-time PCR allows for rapid detection of B. anthracis spores and cells in samples based
simply on the presence of DNA. However, since the DNA from dead spores and cells can also be
detected by this method, a positive sample result does not confirm the presence of viable spores
or cells. Therefore, this method is usually used for a time- and cost-effective presumptive
analysis of samples. This section includes real-time PCR method with appropriate sample
processing procedures for detection of B. anthracis spores.
Acceptable sample types: Gauze wipes (2” × 2” 50% rayon/50% polyester [Kendall™
Versalon™ Cat. No. 8042 or equivalent]), air filters (37 mm Fluoropore™ [Millipore® Cat. No.
FSLW04700 or equivalent]), swabs (macrofoam [VWR Cat. No. 89022-994 small swabs or
89022-984 extra-large swabs or equivalent]), Sponge-Stick sampling tools (3M Inc. Cat. No.
SSL100 or equivalent), 37-mm mixed cellulose ester (MCE) filter cassettes (SKC Cat. No. 225-
9543 or equivalent), vacuum filters (3M Forensic, Precision Data Products Cat. No. FF-1 with 4”
diameter filter or equivalent), drinking water and decontamination waste water
9.1 Sample Processing: Spore Recovery
Note: All subsequent procedures involving manipulation of samples must be carried out in a BSC
using appropriate PPE. The CDC requires BSL-3 handling of this organism. All wastes
should be handled according to CDC & BMBL waste management and disposal requirements.
Prepare monofilament polyester mesh (Section 5.3.3) supports by cutting 2” × 2” squares using
sterile scissors and place squares into a clean ziplock bag. Since the supports are not sterilized
prior to use, ensure that the working surface has been disinfected and sterile gloves are worn
during the process.
Fill sample tube rack with 30 mL or 50 mL screw cap conical tubes, as appropriate. All sample
types (except water samples) may be placed behind a mesh support in the tube to prevent
interference from pipetting activities and to improve efficiency of spore extraction during
vortexing. Using two pairs of sterile forceps, coil the mesh support and then grasp both ends of
the coil with one pair of forceps. Place the support into the tube by holding the sample to the side
of the tube with one pair of sterile forceps and placing the coiled mesh support on top with the
other set of forceps. Figure 1 identifies the appropriate sub-sections for sample processing and
spore recovery for each sample type/tool.
18
Wipes/Air
Filters
9.1.1
9.1.7 9.29.1.20
Figure 1. Sample processing and spore recovery steps for real-time PCR.
9.1.1 Wipe and Air Filter Samples
Place mesh support over wipe or air filter samples in 30 or 50 mL tube by holding the
wipe or air filter to the side of the tube with sterile forceps and placing the coiled mesh
support on top as described in Section 9.1. Ensure the sample and mesh are in the bottom
half of the tube (avoiding the conical portion). Decontaminate workspace with 10% pH
amended bleach (Section 6.20) or bleach wipes (Section 5.1.3) and don a fresh pair of
gloves in between samples. Repeat process for each sample. The support keeps the wipe
or air filter from interfering with pipetting activities and also improves efficiency of spore
extraction during vortexing. Discard all waste in an autoclavable biohazard bag and
decontaminate workspace and equipment with a 10% pH amended bleach solution
(Section 6.20) or bleach wipes (Section 5.1.3). Proceed to Section 9.1.7.
9.1.2 Vacuum Samples (37-mm filter Cassettes and Filters)
Note: All subsequent procedures involving manipulation of 37-mm filter cassettes must be
carried out in a BSC using appropriate PPE.
9.1.2.1 37-mm filter Cassettes
For each 37-mm filter cassette, prepare one 15 mL conical tube containing 11 mL of
sterile Extraction Buffer with Tween and 30% (Section 6.4) and label one 2 oz.
sterile cup (Section 5.1.34). In the BSC remove the conical tube containing the
nozzle and the cassette from the containment bags and wipe the outside of the conical
tube with a disinfectant and place it into a rack. Aseptically add 5 mL of buffer (from
the 11 ml of a pre-measured aliquot of Extraction Buffer with Tween and 30%) to the
conical tube containing the nozzle and tubing and set aside. Remove the band from
around the cassette using a sterile scalpel or sterile pair of scissors. Wipe each
cassette with 10% pH amended bleach solution (Section 6.20) or bleach wipes
(Section 5.1.3) followed by a clean Kimwipe® and discard wipes into an autoclavable
biohazard bag.
Change gloves. Remove the red plug from the front filter side of the cassette; the
plug on the back side should be kept in place. Using a transfer pipette dispense 1 mL
of Extraction Buffer with Tween and 30% from the tube now containing the 6 mL
into the cassette and replace plug. Roll the cassette around to allow the liquid to
touch all surfaces of the inside of the cassette. If there is a large quantity of
particulate matter, more Extraction Buffer with Tween and 30% may be required.
19
Particulate matter should be dampened enough to prevent aerosolization.
Using the cassette tool (Section 5.1.36) pry open the top section of the cassette, using
care not to spill the Extraction Buffer inside the cassette. Set the bottom portion
containing the filter aside carefully (filter side up), and using a transfer pipette rinse
the walls of the cassette with 1-2 mL of Extraction Buffer with Tween and 30%.
Transfer the rinsate using the same pipette to the appropriately labelled 2 oz. sterile
cup (Section 5.1.34). Using the same transfer pipette repeat the rinsing process for
the bottom portion of the cassette and transfer the rinsate to the 2 oz. cup.
Using the cassette tool remove the middle section of the cassette (this piece is
holding the filter in place). Using sterile forceps aseptically remove the filter without
picking up the support filter underneath. Place the filter in the 2 oz. cup with the
rinsates. Use the remainder of the 6 mL Extraction Buffer with Tween and 30% to
rinse walls and bottom section of the cassette and transfer to the 2 oz. cup. Discard
the cassette sections, support filter, plugs, and transfer pipette in an autoclavable
biohazard bag.
Disinfect the outside of the 2 oz. cup with 10% pH amended bleach solution (Section
6.20) or bleach wipes (Section 5.1.3) and place in rack. Decontaminate the BSC with
a 10% pH amended bleach solution (Section 6.20) or bleach wipes (Section 5.1.3)
and don a fresh pair of gloves in between samples in between samples. Repeat
procedure described above for each 37-mm filter cassette.
Seal the conical tubes containing the nozzle and tubing in 5 mL Extraction Buffer
with Tween and 30%, tubing and nozzle with Parafilm®. Place the rack of conical
tubes into the sonicating bath to a level that allows at least 1 inch (~2.5 cm) of tube to
be above the water line. Place a rectangular weight on top of the tubes to prevent
them from floating or tipping over. Sonicate for 1 minute and remove tubes from the
sonicating bath. Dry and disinfect each tube with a 10% amended bleach solution
(Section 6.20) or bleach wipes (Section 5.1.3).
Vortex the conical tubes on high for 2 minutes and transfer the 5 mL suspension to
the appropriate 2 oz. cup.
Seal all of the 2 oz. cups with Parafilm®. Place the rack of 2 oz. cups in the sonicating
bath and cover with a rectangular weight on top of the cups to prevent them from
floating or tipping over. There should be 1 inch (~2.5 cm) between the level of the
water and the cup lids. Sonicate for 3 minutes without heat. Remove rack from the
bath and dry each cup with a Kimwipe® and place in the BSC. Place cups in a
sealable plastic lidded box (Section 5.1.35). Discard all waste in an autoclavable
biohazard bag and decontaminate workspace and equipment with a 10% pH amended
bleach solution (Section 6.20) or bleach wipes (Section 5.1.3).
Using a 10 mL serological pipet, transfer as much suspension as possible from each 2
oz. cup to the corresponding labeled 50 mL conical tubes.
Proceed to Section 9.1.20.
9.1.2.2 Vacuum filters
For vacuum filters, ensure that the exposed filter surface (with debris) is facing up and
carefully cut through the evidence tape with a sterile scalpel in order to remove the top of
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the cartridge. Using a pair of sterile forceps, transfer large pieces of debris into the
appropriate 50 mL tube, then fold filter in half with dirty, exposed filter side in, and then
fold in half again in order to fit it into the 50 mL tube. Place folded filter in bottom half
of tube (avoiding conical portion) and using two pairs of sterile forceps place the coiled
mesh support (Section 9.1) on top of filter. Repeat for each sample. Decontaminate the
BSC with 10% amended bleach (Section 6.20) or bleach wipes (5.1.3) and don a fresh
pair of gloves in between samples. Proceed to Section 9.1.7.
9.1.3 Sponge-Stick Samples
carried out in a BSC using appropriate PPE.
If the Sponge-Stick sponge is not in a Stomacher® bag, holding the plastic handle of
the Sponge-Stick with one gloved hand, carefully remove the sponge using sterile
forceps and aseptically transfer it to a Stomacher® bag. Change forceps between
samples.
Add 90 mL of phosphate buffered saline with Tween® 20 (PBST) to each bag. Set
Stomacher® (Section 5.5.28) to 260 rpm.
Place a bag containing a sample into the Stomacher® (Section 5.5.28) so the sponge
rests evenly between the homogenizer paddles and stomach each sample for 1 minute
at 260 rpm.
Open the door of the Stomacher® (Section 5.5.28) and remove the bag. Grab the
sponge from the outside of the bag with hands. With the bag closed, move the
sponge to the top of the bag while using hands to expel liquid from the sponge.
Open the bag, remove and discard the sponge in an autoclavable biohazard bag, using
sterile forceps.
Repeat steps described above for each sample, changing forceps and gloves between
samples.
Allow bags to sit for 10 minutes to allow elution suspension foam to settle.
Gently mix the elution suspension in the Stomacher® bag up and down three times
with a sterile 50 mL pipet. Remove half of the suspension volume (~45-46 mL) and
place it in a labeled 50 mL screw capped centrifuge tube. Place the remaining
suspension (~45-46 mL) into a second 50 mL tube. Adjust the suspension volumes in
both the tubes to ensure they are similar.
Record suspension volumes on tubes and data sheet.
Process elution suspension for each sample as described above.
Place 50 mL tubes into sealing centrifuge buckets and decontaminate centrifuge
buckets before removing them from the BSC.
Centrifuge tubes at 3500 × g, with the brake off, for 15 minutes in a swinging bucket
rotor.
Note: A higher × g is preferred as long as the speed is within the tube and rotor
specifications.
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Using a sterile 50 mL pipet for each sample, carefully remove ~43-44 mL leaving
approximately 2 mL of the supernatant from each of the two 50 mL tubes and discard
it in an autoclavable biohazard bag. The pellet may be easily disturbed and not
visible, so keep the pipet tip away from the bottom of the tube.
Set the vortexer (Section 5.5.16) to high intensity. Set the sonicator bath (Section
5.5.27) to high.
Vortex the tubes for 30 seconds to resuspend the pellets and transfer the tubes to the
sonicator bath and sonicate for 30 seconds. Repeat the vortex and sonication cycles
two more times.
Note: As an alternative to sonication, tubes may be vortexed for 2 minutes in 10 second
bursts using a multi-tube vortexer.
Remove suspension from one tube with a sterile 5 mL pipet and combine it with the
suspension in the other tube from the same sample. Measure final volume of
suspension with 5 mL pipet and record the result on the tube and data sheet.
Repeat vortexing and sonication steps for each sample.
Discard all waste in an autoclavable biohazard bag and decontaminate workspace and
equipment with a 10% pH amended bleach solution (Section 6.20) or bleach wipes
(Section 5.1.3).
9.1.4 Swab Samples
Note: All subsequent procedures involving manipulation of Swab samples must be carried
out in a BSC using appropriate PPE.
Place swab into the 30 mL tube and cut handle with sterile scissors, if necessary, to fit
into the tube. Using sterile forceps, place the coiled mesh support over the swab (Section
9.1). Repeat process for each sample. Discard all waste in an autoclavable biohazard bag
and decontaminate counters and equipment with a 10% pH amended bleach solution
(Section 6.20) or bleach wipes (Section 5.1.3). Proceed to Section 9.1.7.
9.1.5 Water Samples (Large Volume [10 L – 100 L], Drinking Water)
Please see Appendix A for primary (Section 2.0) and secondary (Section 3.0)
concentration of large volume (10 L-100 L) water samples. For water samples < 10 L
and ≥ 50 mL, please refer to Appendix A, Section 3.0, secondary concentration.
Add 15 mL of sodium phosphate/EDTA/Tween® 20 buffer (Section 6.10) to the 50
mL conical tube with membrane (Appendix A, Section 3.5).
Set vortexer (Section 5.5.16) to high intensity.
Vortex membrane in 10 second bursts for 2 minutes to dislodge spores.
Using sterile forceps remove membrane from the tube and discard in an autoclavable
biohazard bag. Centrifuge suspension at 3500 × g, with the brake off, for 15 minutes
at 4°C.
specifications.
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Remove 12 mL of the supernatant without disturbing/dislodging the pellet; resuspend
the pellet by vortexing in the remaining volume.
Repeat for each sample.
(Section 5.1.3).
Use a 1.5 mL aliquot for DNA extraction using bead-beating, as described in Section
9.2.
9.1.6 Water Samples (Small Volume [< 50 mL], Surface or Drinking Water)
Transfer 30 mL of water sample into a 50 mL screw cap conical tube.
Add 10 mL of sodium phosphate/EDTA/Tween® 20 buffer (Section 6.10) and mix by
vortexing for 30 seconds.
Centrifuge at 3500 × g, with the brake off, for 15 minutes at 4°C.
specifications.
Remove 37 mL of the supernatant without disturbing/dislodging the pellet. The
volume of supernatant remaining should not be below the conical portion of the tube.
Resuspend the pellet by vortexing for 30 seconds in the remaining volume.
Repeat for each sample.
(Section 5.1.3).
Use a 1.5 mL aliquot for DNA extraction using bead-beating, as described in Section
9.2.
9.1.7 Add 20 mL (5 mL for swabs) of cold (4°C) extraction buffer with Tween® 20 (Section
6.4) to environmental samples (Sections 9.1.1, 9.1.2, 9.1.4) placed in 30 mL tubes (50
mL tubes for vacuum filters) in tube rack. Use a new serological pipet to transfer buffer
from a sterile, 250 mL screw capped bottle to each tube (keep bottle cap loosely over
opening between transfers). Uncap one tube at a time, add 20 mL extraction buffer with
Tween® 20, close tube and place it back in tube rack. Repeat for each sample tube.
Check that all caps are on tubes securely. Label tubes, as appropriate, and document
location in rack. If needed, the tube caps can be sealed with Parafilm.
9.1.8 Place tube rack in plastic bag, seal, bleach bag and double bag, prior to transferring to
platform vortexer (outside BSC).
9.1.9 Vortex samples for 20 minutes on platform vortexer (Section 5.5.15), with speed set to 7.
9.1.10 After vortexing, transfer sample tube rack to BSC. Remove tube rack from plastic bag.
9.1.11 Vortex one sample tube on single-tube vortexer (Section 5.5.16), in the BSC, for 3-5
seconds. For samples containing large amounts of debris, let sample sit for 30 seconds to
allow large particles to settle prior to dispensing aliquots.
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9.1.12 Open the tube. Using a 25 mL serological pipet, transfer as much liquid volume as
possible (while avoiding settled particles) to a fresh appropriately labeled 50 mL conical
tube. Discard pipets in an autoclavable biohazard waste container. Cap sample tube and
place tube back in rack. Change gloves.
9.1.13 Repeat Sections 9.1.11-9.1.12 for each sample tube.
9.1.14 Perform second spore extraction. Uncap one sample tube at a time.
9.1.15 Add 14 mL (5 mL for swabs) of cold (4°C) extraction buffer without Tween® 20 (Section
6.5) to each sample tube, one at a time, with a new 25 mL serological pipet and a fresh
pair of gloves for each sample. Keep buffer bottle loosely covered between transfers.
Recap sample tube after buffer addition.
9.1.16 After adding extraction buffer to all tubes, check that all caps are on securely. Place tube
rack in plastic bag, seal, bleach bag and double bag, prior to transferring to platform
vortexer (outside BSC).
9.1.17 Vortex rack for 10 minutes on platform vortexer (Section 5.5.15), with speed set to 7.
9.1.18 Repeat Sections 9.1.10-9.1.11.
9.1.19 Open the tube. Using a 25 mL serological pipet, transfer as much liquid volume as
possible (while avoiding settled particles) to the original 50 mL tube containing the first
extraction suspension to combine the extracts. Discard pipets in an autoclavable
biohazard waste container. Cap sample tube and place tube back in rack. Change gloves.
9.1.20 Centrifuge the 50 mL conical tubes containing the suspension at 3500 × g, with the brake
off, for 15 minutes at 4°C.
specifications.
9.1.21 Leaving approximately 3 mL in the tube, carefully discard the supernatant in an
autoclavable biohazard bag, using a serological pipet without disturbing/dislodging the
pellet. Ensure that the volume of liquid remaining is not below the conical portion of the
tube. Resuspend the pellet by vortexing.
9.1.22 Add 25 mL (10 mL for swabs) of sodium phosphate/EDTA/Tween® 20 buffer (Section
6.10), tightly cap the tube, and mix the suspension by vortexing.
9.1.23 Repeat Sections 9.1.21-9.1.22 for each sample.
9.1.24 Centrifuge the suspension at 3500 × g, with the brake off, for 15 minutes at 4°C.
specifications.
9.1.25 Carefully discard 25 mL (10 mL for swabs) of supernatant in an autoclavable biohazard
bag without disturbing/dislodging the pellet. Resuspend the pellet by vortexing in the
remaining volume. Repeat process for each sample.
9.1.26 Discard all waste in an autoclavable biohazard bag and decontaminate workspace and
(Section 5.1.3).
9.1.27 Use a 1.5 mL aliquot for DNA extraction using bead-beating, as described in Section 9.2.
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Note: Alternate DNA extraction-purification procedures may be used (e.g., MagNA-Pure LC
instrument).
9.2.1 In a clean room, using the 8 cap strips, transfer two level capfuls (~100 mg) of the 106
µm glass beads and two level capfuls (~100 mg) of the 425-600 µm glass beads (using a
clean strip of caps between bead sizes) into each gasketed, capped 2 mL bead-beating
tube.
9.2.2 In the BSC, pipet 1.0 mL of the suspension (sample eluent, Section 9.1.24) into pre-
labeled, gasketed, capped bead-beating 2 mL tube containing glass beads. Replace cap
on tube securely. Wipe outside of tube with a 10% pH amended bleach solution (Section
6.20) or bleach wipes (Section 5.1.3). Store the remaining suspension at 4ºC.
9.2.3 Insert tubes in tube holders of the bead-beater (Section 5.5.25) and set the timer for 3
minutes (180 seconds). Bead-beat at 3450 oscillations/minute to disrupt spores to release
the DNA.
9.2.4 Remove tubes from bead-beater (tubes will be warm), and place in a cold block for 2
minutes (or until cool to touch). If any tubes leak during bead-beating, wipe tubes and
bead-beater thoroughly with a 10% pH amended bleach solution (Section 6.20) or bleach
wipes (Section 5.1.3).
To remove potential PCR inhibitors in very dirty samples, in the BSC centrifuge the
bead-beating tubes at 7000 rpm for 2 minutes in a microcentrifuge using a fixed angle
rotor to pellet beads and particulate matter. Using a micropipettor, carefully remove 1.0
mL and follow the Manual DNA Extraction and Purification Procedure (Section 10.4).
Then after, proceed to Section 9.2.10 and onwards.
9.2.5 Supernatant Separation and Transfer
Set up tubes; for each sample, label one 1.5 mL microcentrifuge tube, two yellow-top
0.22µm Ultrafree®-MC filter units (Section 5.1.30; Millipore® Cat. No.
UFC30GV0S), one Amicon® Ultra filter insert (Section 5.1.29; Millipore® Cat. No.
UFC503096), and six Amicon® Ultra collection tubes (Section 5.1.29; Millipore®
Cat. No. UFC50VL96) with sample ID for each bead beating tube (Section 9.2.4);
and one 0.1 µm Ultrafree®-MC filter device (Section 5.1.31; Millipore® Cat. No.
UFC30VV00).
Note: It may not be necessary to label all the collection tubes as long as the Amicon Ultra
filter insert is clearly labeled.
In a BSC, centrifuge the bead-beating tubes (Section 9.2.4) at 7000 rpm for 2 minutes
in a microcentrifuge using a fixed angle rotor to pellet beads and particulate matter.
Using a micropipettor, carefully transfer 0.4 mL of the supernatant from the bead-
beating tube to each of the two yellow-top filter units (Section 5.1.30; Millipore® Cat.
No. UFC30GV0S). Avoid beads and particulate matter at bottom of bead-beating
tube). Cap the filter units.
Centrifuge at 7000 revolutions per minute (rpm) for 3 minutes at 4ºC.
Note: Ensure that the supernatant has been filtered. Centrifuge for an additional 2 minutes
if there is any supernatant in the filter.
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Open the filter units; remove the yellow-top filter inserts with sterile disposable
forceps (gripping only on the sides) and discard in an autoclavable biohazard bag.
Transfer 0.4 mL of the filtrate from the collection tubes to Amicon® Ultra filter
inserts (Section 5.1.29; Millipore® Cat. No. UFC503096). Do not transfer any
particulate matter that may be evident at bottom of the tubes. Place filter inserts into
new collection tubes (Section 5.1.29; Millipore® Cat. No. UFC50VL96). Cap the
filter units.
Centrifuge at 7000 rpm for 2 minute at 4ºC.
Open the filter units. Remove the Amicon® Ultra filter inserts (Section 5.1.29;
Millipore® Cat. No. UFC503096) with disposable forceps (gripping only the sides)
and transfer to new collection tubes (Section 5.1.29; Millipore® Cat. No.
UFC50VL96). Dispose of old collection tubes with filtrate in an autoclavable
biohazard bag.
Transfer the remaining (0.4 mL) filtrate from all of the second set of yellow-top filter
units to the corresponding sample Amicon® Ultra filter inserts (Section 5.1.29;
Millipore® Cat. No. UFC503096). Do not transfer any particulate matter that may be
evident at bottom of tubes. Cap the filter units.
Centrifuge at 7000 rpm for 3 minutes at 4ºC.
Open the filter units. Remove the Amicon® Ultra filter inserts using disposable
forceps (gripping only the sides) and transfer to new collection tubes (Section 5.1.29;
Millipore® Cat. No. UFC50VL96). Dispose of old collection tubes with filtrate in an
autoclavable biohazard bag.
9.2.6 First Wash
Add 400 µL of 1X TE buffer (Section 6.11) to the Amicon® Ultra filters. Cap the
filter units.
Open the filter units. Transfer the Amicon® Ultra filter inserts (Section 5.1.29;
Millipore® Cat. No. UFC503096) with disposable forceps (gripping only the sides) to
new collection tubes (Section 5.1.29; Millipore® Cat. No. UFC50VL96). Dispose of
used collection tubes with filtrate in an autoclavable biohazard bag.
9.2.7 Second Wash
Add 400 µL of 1X TE buffer to the Amicon® Ultra filters. Cap the filter units.
9.2.8 Third Wash
Add 400 µL of 1X TE buffer to the Amicon® Ultra filters. Cap the filter units.
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9.2.9 Fourth Wash
Add 400 µL of PCR-grade water (Section 6.9) to the Amicon® Ultra filters. Cap the
filter units.
Centrifuge at 7000 rpm for 1 minute at 4ºC.
Check fluid level in the Amicon® Ultra filter inserts (Section 5.1.29; Millipore® Cat.
No. UFC503096). If fluid level is above 200 μL, pulse spin for about 10 seconds (or
less) until about 100 µL of fluid is retained on top of white base.
If there is less than 100 µL of extract, transfer DNA extract back to the same
Amicon® Ultra filter insert (Section 5.1.29; Millipore® Cat. No. UFC503096) and add
100 µL PCR-grade water and pulse spin to obtain about 100 µL on filter.
Note: Very dirty samples may require additional washes to remove any potential inhibitors.
9.2.10 Filtration of DNA Extract using 0.1 µm Centrifugal Filter Device (Section 5.1.31)
Centrifugal filtration with 0.1-μm Ultrafree®-MC filter device following extraction of
DNA allows for the removal of any B. anthracis spores which may have contaminated
DNA preparations, making the samples safe without compromising the sensitivity of the
real-time PCR assay (Reference 15.8).
Using a micropipettor, carefully remove all of the retentate (~ 100 µL) from the
Amicon® Ultra filter inserts (Section 5.1.29; Millipore® Cat. No. UFC503096) and
transfer to corresponding 0.1 µm Ultrafree®-MC filter devices (Section 5.1.31;
Millipore® Cat. No. UFC30VV00). Do not allow the micropipettor tip to touch the
filter membrane. Avoid transferring any particulate matter that may be evident at
bottom of the tubes. Close the caps. Discard the Amicon® Ultra filter inserts
(Section 5.1.29; Millipore® Cat. No. UFC503096) with collection tubes (Section
5.1.29; Millipore® Cat. No. UFC50VL96) in an autoclavable biohazard bag.
Repeat the above step for all the samples/retentates.
Place the Ultrafree®-MC filter devices (Section 5.1.31; Millipore® Cat. No.
UFC30VV00) into a centrifuge (Section 5.5.7; Eppendorf 5415R/5424R) and balance
the rotor head.
Centrifuge at 8000 &

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